CHARGERIN---ITS TERTIARY STRUCTURE AND THE MECHANISM OF ENERGY TRANSDUCTION

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Recently we purified a hydrophobic protein named chargerin 11 from rat liver mitochondria [Higuti, T., et al. J. Biol. Chem. 263, 6772-6776 (1988)] , which was encoded by the unidentified reading frame URFA6L of mitochondrial DNA. Although chargerin IT is hydrophobic and soluble in a mixture of chloroform and methanol (2:1), it has unbalanced positive charges in its sequence. Chargerin 11 is one of the subunits of Fo of H^+-ATP synthase. Furthermore. an antibody against chargerin II inhibited energy-transduction by H^+-ATP synthase in mitoplasts in an energy-dependent fashion. suggesting that energization of H^+-ATP synthase causes a conformational change in chargerin II [Uchida. J. et al. Biochem. Biophys. Res. Commun. 165, 449-456 (1987)].These unique features of chargerin II suggest that it has an essential role in the energy transduction by mitochondrial ATP synthase, in good accord with the charge-transfer coupling hypothesis [Higuti, T. , Mol. Cell. Biochem. 166, 37-61 (1984)].In … More the present research project. we have obtained the following important findings.1. The orientation of chargerin II in Fo of the ATP synthase of rat liver mitochondria was examined using antibodies against peptides of chargerin II. Results showed that its N-terminal region (about 8 amino acid residues) was exposed on the surface of the C-side of Fo, but its C-terminal and charge-cluster regions were buried in Fo.2. By using the anisotropic inhibitors of energy transduction. which were found by us previously, we found that "an internal electric field" in H^+-transport redox complexes and ATP synthase is formed in their energized state. in good accord with the charge-transfer coupling hypothesis.3. The contents of chargerin If in the H^+-ATP synthase purified from rat liver mitochondria and in submitochondrial particles were determined by radioimmunoassay. Results showed that the H^+-ATP synthase contained chargerin II in a molar ratio of one to one. This is the first report on the stoichiometry of the A6L-product in mitochondrial H^+-ATP synthase.4. The subunits of H^+-ATP synthase from rat liver mitochondria were purified on a reverse-phase column. The sequences of these subunits were determined by the protein sequences. The subunit b and factor 6 were firstly found in rat. Then we synthesized the probe DNA for the subunit b and succeeded to clarify the sequence of cDNA for the import precursor of subunit b. The cDNA of subunit b contained 1,124 base pairs. The sequence contained the coding region of 42 amino acids of the import signal reptide and 214 amino acids of the mature protein. Less